Stable peptide-based furin inhibitors

ABSTRACT

It is provided furin inhibitors and their uses for treating pathogen infection. Particularly, it is provided a method or use for the treatment of a pathogen infection, in a subject, comprising administering to the subject a therapeutically effective amount of the furin inhibitors or the composition disclosed, thereby preventing or treating pathogen infection, in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of U.S. Provisional ApplicationNo. 61/530,478, filed Sep. 2, 2011, which is hereby incorporated hereinby reference in its entirety.

TECHNICAL FIELD

The present description relates to furin inhibitors and their stableanalogues.

BACKGROUND

Pro-protein convertases (PCs) are serine proteases that optimally cleavesubstrates at R-X-K/R-R motif. These processing events, resulting in theactivation of protein precursors, occur at multiple levels of cellsecretory pathways, and even at the cell surface.

In mammalian cells, seven members of this family have been identified:furin, PACE4, PC1/3, PC2, PC4, PC5/6 and PC7, with differentialexpression in tissues, ranging from ubiquitous (eg. furin) to anendocrine restricted expression (PC1/3 and PC2).

In addition to normal cell functions, PCs, including furin, areimplicated in many pathogenic states, because they process to maturitymembrane fusion proteins and pro-toxins of a variety of bacteria andviruses, including Shiga toxin, anthrax, botulinum toxins, influenza AH₅N₁ (bird flu), flaviviruses, Marburg and Ebola viruses (Thomas, 2002,Nat. Rev. Mol. Cell. Biol., 3: 753-766). After processing by furin andthe subsequent endocytic internalization in the complex with therespective cell surface receptor followed by acidification of theendosomal compartment, the processed, partially denatured, infectiousproteins expose their membrane-penetrating peptide region and escapeinto the cytoplasm (Collier and Young, 2003, Annu. Rev. Cell Dev. Biol.,19: 45-70). Pathogens or their toxins, including influenza virus,Pseudomonas, Shiga toxin and anthrax toxins, require processing by hostproprotein convertases (PCs) to enter host cells and to cause disease.

Furin is a widespread and indispensable protease active in both thesecretory pathway and on, or near the cell surface. Furin is produced asa proprotein, cycles between the Golgi apparatus, endosomes, and cellmembrane. Furin is active in both embryogenesis and in mature cells. Atsteady state, furin is localized principally in the trans-Golgi network(TGN)/Endosomal system. Depending upon its location in the system, furincatalyses a number of different reactions, all involving proteolyticcleavage of proproteins. For example, in the TGN/biosynthetic pathwayfurin cleaves a propeptide to give active pro-P nerve growth factor(pro-p-NGF). Similarly, furin cleaves propeptides thereby activatingpro-bone morphogenic protein-4 (pro-BMP-4) and the “single-chain”insulin pro-hormone to form the higher activity latter-form entity.

Furin cleaves proteins just downstream of a basic amino acid targetsequence (canonically, Arg-X-(Arg/Lys)-Arg′). A number of pathogensexploit host cell furin activity to help activate proteins involved inpathology. For example host cell furin cleaves the Ebola Zairepro-glycoprotein (pro-GP) protein as part of the virus's infectiouscycle. Further, the envelope proteins of viruses such as HIV, influenzaand dengue fever viruses must be cleaved by furin or furin-likeproteases to become fully functional. Anthrax toxin, pseudomonasexotoxin, Shiga toxin (Garred et al., 1995, J Biol Chem, 270:10817-10821) and papillomaviruses must be processed by furin duringtheir initial entry into host cells.

Additionally, furin in the early endosome, cleaves propeptides toproduce active bacterial proteins such as diptheria toxins, shigalatoxin, shigala-like toxin 1, and Pseudomonas exotoxin A. Furin processesthe coat protein of Human Immunodeficiency Virus (HIV) and PA toxinproduced by Bacillus anthrasis.

Additional pathogen derived propeptides processed by furin and othersubtilisins-like proteases include, for example, viral proteins such asHIV-1 coat protein gp160, and influenza virus haemagglutinin as well asbacterial proteins such as diphtheria toxin, and anthrax toxin (seeDecroly et al., 1194, J. Biol. Chem., 269: 12240-12247; and Vey et al.,1994, Cell. Biol., 127: 1829-1842).

In view of its large spectra of activity, furin has become the focus ofconsiderable study and identifying inhibitors of furin are underconsideration as therapeutic agents for treating pathogenic infection.

PCT application publication No. WO 2009/023306, which is herebyincorporated by reference in its entirety, discloses furin inhibitorsand their uses for limiting pathogenic infections.

There is still a need to be provided with improved furin inhibitors. Itwould be highly desirable to be provided with more stable and selectivefurin inhibitors that are effective in treating pathogen infections.

SUMMARY

One aim of the present description is to provide furin inhibitors andtheir uses for treating pathogen infection.

It is provided a peptide sequence comprising the following formula I:

Z-Xaa₈-Xaa₇-Xaa₆-Xaa₅-Arg₄-Xaa₃-Xaa₂-Arg₁-Xaa_(1′)  (I)

wherein

-   -   Xaa_(1′) is absent or any amino acids, peptidomimetic, or        stereoisomer thereof;    -   Arg₁ and Arg₄ are independently arginine, an analogue, a mimic        of arginine or stereoisomer thereof;    -   Xaa₂ is a basic amino acid, an analogue, or stereoisomer        thereof;    -   Xaa₃ is independently any amino acids, an analogue or        stereoisomer thereof;    -   Xaa₅, Xaa₆, Xaa₇ and Xaa₈ independently are Lys, Arg or His,        peptidomimetic or stereoisomer thereof; and    -   Z comprises at least one of acetyl, azido and PEG group, fatty        acids, steroid derivatives and sugars linked to the N-terminal        of the peptide sequence;    -   with the proviso that Xaa₅, Xaa₆, Xaa₇ and Xaa₈ are not aromatic        or negatively charged amino acids.

The sugars encompassed can be mono or poly sugars.

The term “analogues” is intended to mean analogues of amino acids andpseudo peptide bonds, such as “click”, aza, -ene (double conjugated orunconjugated C═C bonds).

In a particular embodiment, the N terminus of the inhibitor is acylated(e.g. acetylated). Further, the N terminus acylation is with fatty omegaamino acids or with steroid derivatives.

The fatty (saturated or unsaturated) omega amino acids can be C2 to C18,more preferably the fatty omega amino acids are selected from the groupconsisting of 11-amino undecanoic acid or 8-amino octanoic acid.

According to another aspect of the present description, there isprovided a composition comprising furin inhibitors as defined herein anda carrier.

In another embodiment, the composition further comprises at least oneanti-viral drug.

Concurrent administration” and “concurrently administering” as usedherein includes administering a composition as described herein and oneanti-viral drug compound in admixture, such as, for example, in apharmaceutical composition, or as separate formulation, such as, forexample, separate pharmaceutical compositions administeredconsecutively, simultaneously, or at different times.

Preferably, the composition is adapted for delivery by at least one ofthe following route selected from the group consisting of oral, mucosal,intranasal, intraocular, intratracheal, intrabronchial, intrapleural,intraperitoneal, intracranial, intramuscular, intravenous,intraarterial, intralymphatic, subcutaneous, intratumoral, gastric,enteral, colonic, rectal, urethral and intravesical route.

According to still another aspect of the present invention, there isprovided a method of lowering furin activity in a cell, comprisingcontacting the furin inhibitors or the composition as defined hereinwith the cell, thereby lowering furin activity in the cell.

According to yet another aspect of the present description, there isprovided a method of reducing pathogen proliferation in a subject,comprising administering the furin inhibitors or the composition asdefined herein to the subject, thereby reducing the proliferation of thepathogen in the subject.

Pathogen encompassed herein can be a bacterial pathogen such as Anthrax,Pseudomonas, Botulism, Diphtheria, Aeromonas or Shigella; or can be aviral pathogen such as Influenzavirus A, parainfluenza, Sindbis virus,Newcastle disease virus, flavivirus, cytomegalovirus, herpesvirus, HIV,Measles virus, infectious bronchitis virus, Coronavirus, Marburg virus,Ebola virus or Epstein-Barr virus.

According to yet a further aspect of the present description, there isprovided a method for the treatment of pathogen infection in a subject,comprising administering to said subject a therapeutically effectiveamount of the furin inhibitors or the composition as defined herein,thereby preventing or treating pathogen infection in the subject.

Preferably, the cell is in a subject. More preferably, the cell hasincreased furin activity.

According to still a further aspect of the present description, there isprovided the use of the furin inhibitors or the composition as definedherein in the manufacture of a medicament for treating pathogeninfection in a subject.

According to yet another aspect of the present description there isprovided the use of the furin inhibitors or the composition as definedherein for lowering furin activity in a cell, for reducing proliferationof a pathogen in a subject.

The terms used herein are explained below. Each term, alone or incombination with another term, means as follows.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched and comprising about 1 to about 20 carbon atoms in the chain.Preferred alkyl groups contain about 1 to about 12 carbon atoms in thechain. More preferred alkyl groups contain about 1 to about 6 carbonatoms in the chain. Branched means that one or more lower alkyl groupssuch as methyl, ethyl or propyl, are attached to a linear alkyl chain.“Lower alkyl” means a group having about 1 to about 6 carbon atoms inthe chain which may be straight or branched. Non-limiting examples ofsuitable alkyl groups include methyl, ethyl, n-propyl, isopropyl andt-butyl.

The terms “alkenyl” represent a linear, branched or cyclic aliphatichydrocarbon group which may be straight or branched and comprising about1 to about 20 carbon atoms and has one or more double bonds in thechain.

“Alkylene” means a difunctional group obtained by removal of a hydrogenatom from an alkyl group that is defined above. Non-limiting examples ofalkylene include methylene, ethylene and propylene.

“Aryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 6 to about 14 carbon atoms, preferably about 6 to about10 carbon atoms. Examples include but are not limited to phenyl, tolyl,dimethylphenyl, fluoenryl, aminophenyl, anilinyl, naphthyl, anthryl,phenanthryl or biphenyl.

“Arylene” means a difunctional group obtained by removal of a hydrogenatom from an aryl group that is defined above. Examples include but arenot limited to phenylene, tolylene, dimethylphenylene, fluorene,aminophenylene, anilinylene, naphthylene, anthrylene, phenanthrylene orbiphenylene.

“Heteroaryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 5 to about 14 ring atoms, preferably about 5 to about10 ring atoms, in which one or more of the ring atoms is an elementother than carbon, for example nitrogen, oxygen or sulfur, alone or incombination. The prefix aza, oxa or thia before the heteroaryl root namemeans that at least a nitrogen, oxygen or sulfur atom respectively, ispresent as a ring atom. A nitrogen atom of a heteroaryl can beoptionally oxidized to the corresponding N-oxide. Non-limiting examplesof suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl,pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl,isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl,pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl,quinoxalinyl, phthalazinyl, oxindolyl, imidazo[1,2-a]pyridinyl,imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl,benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl,quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl,isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, benzothiazolyl and thelike.

“Heteroarylene” means a difunctional group obtained by removal of ahydrogen atom from a heteroaryl group that is defined above.Non-limiting examples of pyridylene, pyrazinylene, furanylene,thienylene and pyrimidinylene.

The term “arylalkylene” represents an aryl group attached to theadjacent atom by an alkylene.

The term “arylalkenylene” represents an aryl group attached to theadjacent atom by an alkenylene.

The term “NR1-alkylene” represents a NR1 group attached to an alkylene.

The term “NR1-alkenylene” represents a NR1 group attached to analkenylene.

The term “NR1-arylene” represents a NR1 group attached to an arylene.

The term “NR1-heteroarylene” represents a NR1 group attached to aheteroarylene.

The term “NR1-arylalkylene” represents a NR1 group attached to anarylalkylene.

The term “NR1-arylalkenylene” represents a NR1 group attached to anarylalkenylene.

The term “alkylene-NR2” represents an alkylene attached to the adjacentatom by a NR2 group.

The term “alkenylene-NR2” represents an alkenylene attached to theadjacent atom by a NR2 group.

The term “arylene-NR2” represents an arylene attached to the adjacentatom by a NR2 group.

The term “heteroarylene-NR2” represents a heteroarylene attached to theadjacent atom by a NR2 group.

The term “arylalkylene-NR2” represents an arylalkylene attached to theadjacent atom by a NR2 group.

The term “arylalkenylene-NR2” represents an arylalkenylene attached tothe adjacent atom by a NR2 group.

The term “NR1-alkylene-NR2” represents a NR1 group attached to analkylene, the alkylene is attached to the adjacent atom by a NR2 group.

The term “NR1-alkenylene-NR2” represents a NR1 group attached to analkenylene, the alkenylene is attached to the adjacent atom by a NR2group.

The term “NR1-arylene-NR2” represents a NR1 group attached to anarylene, the arylene is attached to the adjacent atom by a NR2 group.

The term “NR1-heteroarylene-NR2” represents a NR1 group attached to aheteroarylene, the heteroarylene is attached to the adjacent atom by aNR2 group.

The term “NR1-arylalkylene-NR2” represents a NR1 group attached to anarylalkylene, the arylalkylene is attached to the adjacent atom by a NR2group.

The term “NR1-arylalkenylene-NR2” represents a NR1 group attached to anarylalkenylene, the arylalkenylene is attached to the adjacent atom by aNR2 group.

The terms “alkylene-COOH”, “alkenylene-COOH”, “arylene-COOH”,“heteroarylene-COOH”, “arylalkylene-COOH”, “heteroarylalkylene-COOH” or“alkenyl-COOH” represents an alkylene, an alkenylene, an arylene, aheteroarylene, an arylalkylene, a heteroarylalkylene or an alkenylattached to the adjacent atom by a —COOH group.

The term “independently” means that a substituent can be the same or adifferent definition for each item.

“PEG” means a polyethylene glycol prepared through polymerization ofethylene oxide that are commercially available, and can include a widerange of molecular weights from 300 g/mol to 10,000,000 g/mol.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings.

FIG. 1 illustrates peptidomimetic modifications used in the synthesis ofRXXT variants.

FIG. 2 illustrates the degradation kinetic of the RXXR peptides.

FIG. 3 illustrates the stability measured for RXXT variants, where in(A) plasmatic half-life curves of peptides (RxxT: L0;RxxT[Azaβ₃R]^(P8):L22; Rxx-[AMBA]^(P1): L8; and RxxT[Azaβ₃R]^(P1-P8):L29) in murin plasma treated ex vivo at 37° C. are showed; and in (B)degradation pattern of the different peptides tested in DU145 culturemedia is illustrated.

FIG. 4 illustrates the ability of the RXXT compound to penetrate cellswherein in (A) a FTU-6Ala-RXXT compound was used, compared in (B) to itsvariant coupled to a lipid amino-octanoyl group (Aoc).

FIG. 5 illustrates the absence of toxicity observed for the compoundstested. Toxicity is measured by the release of LDH by cells, LDH being amitochondrial enzyme release during lyses of cells.

FIG. 6 illustrates the calculation method for the IC₅₀ for the generalPC inhibitor dec-RTKR-CMK on the relative cytotoxicity of theEscherichia coli O157:H7 Shiga toxin. The cytotoxicity was measured bythe release of LDH by VERO cells incubated with Shiga toxin preparationdiluted 1:2 and 1:100 (gray and black curves, respectively). Theaddition of the inhibitor lowers the relative cytotoxicity induced bythe toxin in a dose-dependent manner (IC₅₀=11.5 μM), confirming the roleof PCs in this phenomenon.

FIG. 7 illustrates the capability of the tested compounds to inhibitcell fusion (relative to the no treatment control). The furin inhibitorswere used at a single concentration of 100 μM with cell line expressingthe hemagglutinin H5. For comparison purposes, the general PC inhibitordec-RTKR-CMK was also used. (Aoc:lipid amino-octanoyl group, PEGS:polyethylene glycol group).

FIG. 8 schematizes the cell fusion pharmacological assay. In the absenceof the furin inhibitor, fusion of the HA-positive and GFP-positive celllines leads to the emission of fluorescence. In the presence of theinhibitor, the normal cleavage of hemagglutinin (HA) membrane proteindoes not occur, avoiding the cell fusion and the emission offluorescence.

DETAILED DESCRIPTION

It is provided herein furin inhibitors and their uses for treatingpathogen infection.

One of the keys to the development of potent and selective PC inhibitorsis an understanding of the substrate-binding pocket. The deepest regionof the substrate-binding pocket accommodates the consensus motif RXKR(P₄-P₃-P₂-P₁) nearly identical in all PCs. Potency and selectivity aredetermined by a less deeper region that interacts with P₅-P₇-P₆-P₅ ofthe inhibitor peptide (see Henrich et al., 2005, J. Mol. Biol., 345:211-227; Fugere and Day, 2005, Trends Pharmacol. Sci., 26: 294-301;Henrich et al., 2003, Nat. Struct. Biol., 10: 520-526).

Endogenous inhibitors are often a good starting point in the developmentof pharmacological compounds. For example, proSAAS and the 7B2C-terminal peptide are two endogenous inhibitors identified that inhibitPC1/3 and PC2, respectively. PC pro-domains are autoprocessed in cis bytheir cognate PC, but remain bound to the active site through theirC-terminal PC-recognition sequence until the complex reaches thecompartment of zymogen activation. Thus, pro-domains are dual-functionmolecules, being the first substrate and first inhibitor encountered byPCs in cells.

A series of peptide inhibitors with varying degrees of selectivity andpotency for various PCs are known in the art. One particular compoundstand out: RARRRKKRT (or “RXXT compound”) (see WO 2009/023306).

In order to potentially inhibit the effects of furin in for examplepathogenic infections, improved selective inhibitors were prepared andtested from the RXXT compounds.

The nomenclature used to identify amino acid positions in the inhibitorsdisclosed herein is as follows:

Peptide RXXT Ac-R-A-R-R-R-K-K-R-T-NH₂ Ac-P8-P7-P6-P5-P4-P3-P2-P1-P1-NH₂

Structural modifications illustrated in FIG. 1 were made to the RXXTpeptide in order to identify improved inhibitors of furin. The RXXTpeptide was acetylated and amidated in order to improve its stabilityagainst degradation by cellular peptidases. Multiples variants withimproved plasmatic half-life and stability of the RXXT peptide weresynthesized and tested, as listed in Table 1.

TABLE 1 RXXT variants synthesized and tested

Preliminary observation showed that RXXT compounds are more susceptibleto C-terminal degradation (see FIG. 2). Modified variant at theN-terminal part and/or C-terminal part were tested for their cellularstability and plasmatic half-life. As seen in FIG. 3A, N-terminalvariant had no increase in stability. However, RXXT variants modified attheir C-terminal part showed a 2 to 5 increase in their half-life.

The addition of the lipid amino-octanoyl group increased the penetrationof the RXXT peptide (FIG. 4), thus confirming that further modifying thefurin inhibitors with the lipid amino-octanoyl group will increase theirpotency by increasing the penetration of the compound in targeted cell.To demonstrate that the pharmacological activity of the furin inhibitorssynthesized are not due to an increase in toxicity, a LDH release assayhave been performed (FIG. 5).

As a proof of concept, the ability of the general PC inhibitordec-RTKR-CMK to inhibit the toxicity of the Shiga toxin of Escherichiacoli O157:H7 was measured (FIG. 6). After entering a cell via amacropinosome, the Shiga toxin functions as an N-glycosidase, cleaving aspecific adenine nucleobase from the 28S RNA of the 60S subunit of theribosome, thereby halting protein synthesis within the target cells. TheShiga toxin is associated with multiple pathalogies, such as hemolyticuremic syndrome, thrombotic thrombocytopenic purpura, and hemorrhagiccolitis. The Shiga toxin infection is furin-dependent.

As seen in Table 2, the IC₅₀ for the RXXT inhibitors herein and thegeneral PC inhibitor dec-RTKR-CMK were measured. The IC₅₀ for thedec-RTKR-CMK inhibitor was similar to that of peptidesAoc-RxxT[Azaβ₃R]^(P1) and RxxT[Azaβ₃R]^(P1-P8), showing the potency ofthe stabilized peptides described herein to inhibit the furin-dependentinfection by the Shiga toxin of Escherichia coli O157:H7. Other RXXtinhibitors also showed low IC₅₀ values, including Rxx[ΔCO2]^(P1) andRxxT[Azaβ₃R]^(P1). It is known that other bacterial toxins, such asPseudomonas and anthrax, use similar furin-dependent mechanism ofinfection.

TABLE 2 IC₅₀ (μM) measured for the tested furin inhibitors Furininhibitor IC₅₀ (μM) DEC-RTKR-CMK 11.5 RxxT >1000 Aoc-RxxT >1000Rxx[ΔAR-CO2]^(P1) 71.0 Rxx[AMBA]^(P1) >1000 Aoc-Rxx[AMBA]^(P1) N/ARxxT[Azaβ₃R]^(P1) 314.5 Aoc-RxxT[Azaβ₃R]^(P1) 16.7RxxT[Azaβ₃R]^(P8) >1000 Aoc-RxxT[Azaβ₃R]^(P8) >1000 RxxT[Azaβ₃R]^(P1-P8)20.8

As illustrated in FIG. 8, a cellular fusion assay was developed. On thefirst day, a fixed number of HEK293 cells were seeded and transfectedthe next day with the appropriate expression plasmid to generateHA-positive cell line (or activator cell line) and GFP or luciferasepositive cell line (or reporter cell line). For the HA-positive cellline, the transfected plasmid consisted to those constructs at therespective ratio 2.5:0.6:1; pCDNA3.1 containing HA from H5N1 Vietnam2004:pCDNA 3.1 containing N1 from H5N1 Vietnam 2004:pLVX-Tet off vectorcontaining the TAT activator. For the GFP-positive cell line, thetransfected plasmid was either TAT inducible pLVX-Tight-GFP orpLVX-Tight-Luc that expresses GFP or Luciferase reporter gene inpresence of the TAT inducer. The next day, the cells were counted andseeded in a 24 or 96 wells plate at a ratio of 3:1 of HA cell line toGFP cell line. Cell were allowed to adhere and then were treated 36 hwith furin inhibitors in medium containing 1% FBS. Cells were finallytreated to induce cell fusion by titration of the medium with pH3citrate solution to a final pH of 5 for 15 minutes. After 15 min, mediumwas neutralized to physiological pH with HEPES/Bicarbonate medium,allowed to recover and incubated with fresh medium. Reporter gene wasallowed to developed 48 h and then the cells were read for eitherfluorescence (GFP) or Luciferase activity in a multiwells reader.Inhibition of HA cleavage by furin resulted in a decreased fusionbetween gene and the subsequent non-expression of the reporter gene.

Using the cellular fusion assay, it is demonstrated that the stabilizedpeptides described herein have increased potency in inhibiting furinactivity (see FIG. 7). When compared to the RxxT control peptide, sevenpeptides tested have increased potency against furin, namely Aoc-RxxT,Rxx[Δ-CO2]^(P1), Aoc-Rxx[AMBA]^(P1), PEG8-Rxx[AMBA]^(P1),RxxT[Azaβ₃R]^(P8), Aoc-RxxT[Azaβ₃R]^(P8) and RxxT[Azaβ₃R]^(P1-P8).

It is encompassed herein a composition comprising the furin inhibitorsdescribed herein and a carrier.

In accordance with the present description, a carrier or “pharmaceuticalcarrier” is a pharmaceutically acceptable solvent, suspending agent orany other pharmacologically inert vehicle for delivering one or moreactive compounds to an animal, and is typically liquid or solid. Apharmaceutical carrier is generally selected to provide for the desiredbulk, consistency, etc., when combined with components of a givenpharmaceutical composition, in view of the intended administration mode.Typical pharmaceutical carriers include, but are not limited to bindingagents (e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and othersugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate,ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); andwetting agents (e.g., sodium lauryl sulphate, etc.).

In another embodiment, the composition further comprises at least oneanti-viral drug. Concurrent administration” and “concurrentlyadministering” as used herein includes administering a composition asdescribed herein and insulin and/or a hypoglycemic compound inadmixture, such as, for example, in a pharmaceutical composition, or asseparate formulation, such as, for example, separate pharmaceuticalcompositions administered consecutively, simultaneously, or at differenttimes.

The composition can be adapted for delivery by at least one of thefollowing route selected from the group consisting of oral, mucosal,intranasal, intraocular, intratracheal, intrabronchial, intrapleural,intraperitoneal, intracranial, intramuscular, intravenous,intraarterial, intralymphatic, subcutaneous, intratumoral, gastric,enteral, colonic, rectal, urethral and intravesical route.

There is provided a method of reducing pathogen proliferation in asubject, comprising administering the furin inhibitors or thecomposition as defined herein to the subject, thereby reducing theproliferation of the pathogen in the subject.

Pathogen encompassed herein can be a bacterial pathogen such as Anthrax,Pseudomonas, Botulism, Diphtheria, Aeromonas or Shigella; or can be aviral pathogen such as Influenzavirus A, parainfluenza, Sindbis virus,Newcastle disease virus, flavivirus, cytomegalovirus, herpesvirus, HIV,Measles virus, infectious bronchitis virus, Coronavirus, Marburg virus,Ebola virus or Epstein-Barr virus.

Thus, it is provided a method for the treatment of a pathogen infectionin a subject, comprising administering to said subject a therapeuticallyeffective amount of the furin inhibitors or the composition as definedherein, thereby treating pathogen infection in the subject.

Preferably, the cell is in a subject. More preferably, the cell hasincreased furin activity.

There is also provided the use of the furin inhibitors or thecomposition as defined herein in the manufacture of a medicament forpreventing or treating pathogen infection, in a subject.

The furin inhibitors or the composition as defined herein lower orinhibit furin activity in a cell, reducing proliferation of a pathogenin a subject.

The present disclosure will be more readily understood by referring tothe following examples which are given to illustrate embodiments ratherthan to limit its scope.

Example I Preparation of the Furin Inhibitors

The compounds of the present disclosure can be prepared according to theprocedures denoted in the following reaction Schemes and Examples ormodifications thereof using readily available starting materials,reagents, and conventional procedures or variations thereof well-knownto a practitioner of ordinary skill in the art of synthetic organicchemistry. Specific definitions of variables in the Schemes are givenfor illustrative purposes only and are not intended to limit theprocedures described.

Scheme 1: General synthesis of Synthesis of Fmoc-α-methyl-L-Arg(Boc)₂-OH

The synthesis of Fmoc-α-methyl-L-Arg(Boc)₂-OH was performed byguanidinylation of commercially available Fmoc-α-methyl-L-Orn-OH.

The synthesised Fmoc-α-methyl-L-Arg(Boc)₂-OH was used in peptidesynthesis to generate an α-methyl-L-Arg peptide analogues. Asillustrated in scheme 2, the 2-chlorotrityl resin is used for synthesisof protected peptide for further amidation on C-terminus with AMBA orother amines.

As illustrated in scheme 3, the hydrazine resin is used for the solidphase synthesis of protected peptide, where amidation of peptide occurson C-terminus.

The following examples are given only to illustrate the invention andshould not be regarded as constituting any limitation of the scope ofthe invention in its broadest meaning.

Example 1 Synthesis of Aza-β-Arginine Amino Acid Step 1: Protection ofthe Amine

3,3-diethoxypropan-1-amine (1) (5.24 g, 35.6 mmol) was diluted in amixture of dichloromethane (80 mL) and triethylamine (5.12 mL). Thesolution was cooled to 0° C., and a solution ofdi-tert-butyl-dicarbonate (7.85 g, 35.97 mmol) in dichloromethane (20mL) was slowly added over a 15-minute period. The mixture was stirredfor 16 h, at room temperature. The organic phase was washed with 1N HCl(1×100 mL), 0.5N HCl (1×100 mL), and brine (2×100 mL) before it wasdried with anhydrous magnesium sulphate, filtered and concentrated. Lecrude product was purified by flash chromatography on silica gel(eluent: hexanes/ethyl acetate 7:3). The protected amine was obtained asa yellow oil (8.36 g, 94%). ¹H NMR (300 MHz, CDCl₃) δ (ppm) 4.93 (s,1H), 4.53 (t, 1H, J=5.5 Hz), 3.56 (d-quint, 4H, J=38.9 Hz, J=2.3 Hz),3.20 (q, 2H, J=6.2 Hz), 1.79 (q, 2H, J=6.2 Hz), 1.42 (s, 9H), 1.19 (t,6H, J=7.1 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm) 155.9, 101.9, 78.9.61.5, 36.7, 33.4, 28.4, 15.3. IR (CHCl₃) v (cm⁻¹) 3363 (br), 2979, 2920,2880, 1708, 1514, 1448, 1393, 1365, 1171, 1139, 1065.

Step 2: Preparation of the Aldehyde

The protected amine (2) (3.27 g, 13.2 mmol) was diluted in a mixture ofacetic acid (5.1 mL) and water (1.4 mL), and the solution was stirredfor 16 h, at room temperature. The pH of the solution was then slowlybrought up to 7 with solid sodium carbonate. Diethyl ether (15 mL) wasthen added, and the organic phase was washed with water (1×10 mL) andbrine (1×10 mL). After separation, the organic phase was dried withanhydrous magnesium sulphate, filtered and concentrated under reducedpressure. The crude product was quickly purified by flash chromatographyon silica gel (eluent: diethyl ether/pentane 4:6). The aldehyde wasobtained as a yellow oil (530 mg, 23%). ¹H NMR (300 MHz, CDCl₃) δ (ppm)9.74 (s, 1H), 4.97 (s, 1H), 3.46 (t, 2H, J=7.0 Hz), 2.64 (t, 2H, J=7.0Hz), 1.36 (s, 9H).

Step 3: Preparation of the Imine

The aldehyde (3) (530 mg, 3.06 mmol) was diluted in dichloromethane (15mL), and Fmoc-hydrazine (780 mg, 3.06 mmol) was added. The mixture wasstirred for 16 h at room temperature. The solvent was removed underreduced pressure and the crude product was triturated in petroleumether. The imine was obtained as a white powder (730 mg, 68%). ¹H NMR(300 MHz, DMSO-d₅) δ (ppm) 7.87-7.27 (m, 8H), 6.83 (s, 1H), 4.36 (s,2H), 4.22 (s, 1H), 3.29 (s, 2H), 2.23 (s, 2H), 1.32 (s, 9H). ¹³C NMR(75.5 MHz, CDCl₃) δ (ppm) 143.7, 141.3, 127.8, 127.1, 125.0, 120.0,78.4, 67.0, 49.4, 47.2, 38.5, 28.4, 27.7. IR (CHCl₃) v (cm⁻¹) 3344,3255, 3064, 2976, 2927, 1708, 1683, 1531, 1446, 1365, 1248, 1170, 1029.

Step 4: Reduction of the Imine

The imine (4) (730 mg, 1.78 mol) was dissolved in a mixture ofdichloromethane (12 mL) and methanol (8 mL). Sodium cyanoborohydride(146 mg, 62.8 mmol) was added and the pH was slowly brought up to 4 with2N HCl. The mixture was stirred for 45 minutes at room temperature, andthen the pH was brought up to 7 with solid sodium bicarbonate. Themixture was filtered and concentrated under reduced pressure. Theresidue was dissolved in ethyl acetate (30 mL). The organic phase waswashed with water (1×40 mL) and brine (1×40 mL), and then dried withanhydrous magnesium sulphate, filtered and concentrated under reducedpressure. The reduced imine was obtained as a white-orange solid (720mg, 98%). ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 7.86-7.26 (m, 8H), 4.29-4.20(m, 2H), 4.20-4.12 (m, 1H), 2.89 (t, 2H, J=6.3 Hz), 2.70-2.52 (m, 2H),1.43-1.34 (m, 2H), 1.33 (s, 9H). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm)157.0, 156.4, 143.6, 141.3, 127.8, 127.1, 125.0, 120.0, 79.5, 67.3,49.3, 47.1, 38.3, 28.4, 27.4. IR (CHCl₃) v (cm⁻¹) 3318, 3064, 2976,2937, 1700, 1520, 1478, 1450, 1390, 1365, 1273, 1252, 1171, 1040.

Step 5: Preparation of the Benzyl Ester

The reduced imine (5) (720 mg, 1.75 mmol) was dissolved in toluene (22mL) and the mixture was heated to 80° C. Benzyl bromoacetate (521 mg,2.28 mmol) and dried K₂CO₃ (170 mg, 1.23 mmol) were added, and thereaction was stirred for 24 h at 80° C. The mixture was filtered andwashed with ethyl acetate (40 mL). The organic phase was washed withwater (1×30 mL) and brine (1×30 mL) before it was dried with anhydroussodium sulphate, filtered and concentrated under reduced pressure. Thecrude product was purified by flash chromatography on silica gel(eluent: petroleum ether/ethyl acetate 3:1). The benzylic ester wasobtained as a yellow oil (300 mg, 31%). ¹H NMR (300 MHz, CDCl₃) δ (ppm)7.81-7.28 (m, 13H), 5.17 (s, 2H), 4.42 (d, 2H, J=7.2 Hz), 4.19 (s, 1H),3.72 (s, 2H), 3.21 (s, 2H), 2.97 (s, 2H), 1.68-1.54 (m, 2H), 1.42 (s,9H). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm) 169.5, 156.5, 155.9, 143.7,141.4, 128.7, 128.6, 128.4, 127.7, 127.1, 126.0, 120.0, 119.8, 77.2,66.7, 57.2, 54.2, 47.3, 44.5, 38.6, 28.4, 27.4. IR (CHCl₃) v (cm⁻¹)3350, 3064, 2972, 1739, 1729, 1693, 1682, 1609, 1503, 1453, 1390, 1365,1248, 1171, 1107.

Step 6: preparation of N,N-di-(tert-butoxycarbonyl)-guanidine asdescribed in Journal of Organic Chemistry, Vol. 63, No. 23, 1998

Guanidine chlorhydrate (7) (12.3 g, 128 mmol) and sodium hydroxide (20.8g, 519 mmol) were dissolved in water (125 mL), and 1,4-dioxane (250 mL)were added. The mixture was cooled to 0° C. and di-tert-butyl-carbonate(62.9 g, 288 mmol) was added. The mixture was allowed to warm at roomtemperature within 16 h. The solution was concentrated in vacuo toone-third of its initial volume. Water (150 mL) was added to theresulting mixture, and the solution was extracted with ethyl acetate(3×80 mL). The organic phase was then washed with 10% citric acid (1×100mL), water (1×100 mL) and brine (1×100 mL), dried with anhydrousmagnesium sulfate, filtered and concentrated under reduced pressure. Thecrude product was purified by flash chromatography on silica gel(eluent: 100% dichloromethane to dichloromethane/methanol 95:5). Thedi-protected guanidine was obtained as a white powder (30.54 g, 91%). ¹HNMR (300 MHz, DMSO-d₆) δ (ppm) 10.42 (s, 1H), 8.47 (s, 1H), 1.37 (s,18H). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm) 158.3, 82.3, 28.1. IR (CHCl₃) v(cm⁻¹) 3407, 3124, 2976, 2930, 1792, 1641, 1549, 1453, 1397, 1365, 1248,1153.

Step 7: preparation of N,N-di-Boc-N′-trifluoromethanesulfonylguanidineas described in Journal of Organic Chemistry, Vol. 63, No. 23, 1998

Under an inert atmosphere, N,N-di-(tert-butoxycarbonyl)-guanidine (8)(10 g, 38 mmol) was dissolved in anhydrous dichloromethane (200 mL). Themixture was cooled to −78° C., and triethylamine (5.65 mL, 40.5 mmol)was added. Trifluoromethanesulfonic anhydride (6.81 mL, 40.5 mmol) wasadded dropwise, over a 30-minute period. The reaction mixture wasstirred for 16 h at room temperature. The solution was washed with 2Msodium bisulphate (1×200 mL) and water (1×200 mL), and the organic phasewas dried with anhydrous sodium sulphate, filtered and concentrated. Thecrude product was purified by flash chromatography on silica gel(eluent: 100% dichloromethane) and recrystallized in hexanes.N,N-di-Boc-N′-trifluoromethanesulfonylguanidine was obtained as whitecrystals (11.74 g, 78%). ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 1.43 (s,18H). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm) 151.4, 121.4, 117.1, 86.0, 27.8.IR (CHCl₃) v (cm⁻¹) 3304, 2983, 1785, 1736, 1626, 1556, 1464, 1376,1340, 1259, 1192.

Step 8: addition of guanidine

Benzylic ester (6) (300 mg, 0.54 mmol) was dissolved in dichloromethane(1.65 mL), trifluoroacetic acid (1.65 mL) was added. The mixture wasstirred for 16 h at room temperature. Dichloromethane (15 mL) and water(5 mL) were added, and the pH was slowly brought up to 8 with solidsodium carbonate. After separation, the organic phase was washed withwater (1×40 mL) and brine (1×40 mL), and then dried with anhydrousmagnesium sulphate, filtered and concentrated under reduced pressure tothe half of its volume. Triethylamine (82 μL) andN,N-di-Boc-N′-trifluoro methanesulfonylguanidine (9) (190 mg) were addedand the mixture was stirred on 16 h. The solution was then washed with2M sodium bisulphate (1×10 mL), a saturated solution of sodiumbicarbonate (1×10 mL), water (1×15 mL) and brine (1×15 mL). The organicphase was dried with anhydrous sodium sulphate, filtered andconcentrated. The crude product was purified by flash chromatography onsilica gel (eluent: petroleum ether/ethyl acetate 7:3). The product (10)was obtained as a yellow oil (291 mg, 77%)¹H NMR (300 MHz, CDCl₃) δ(ppm) 7.76-7.11 (m, 13H), 5.16 (s, 2H), 4.41 (s, 2H), 4.21 (s, 1H), 3.79(s, 2H), 3.54 (s, 2H), 2.98 (s, 2H), 1.70 (s, 2H), 1.48 (s, 18H). ¹³CNMR (75.5 MHz, CDCl₃) δ (ppm) 170.5, 163.6, 156.2, 153.2, 143.8, 141.3,135.2, 128.7, 128.6, 128.4, 127.7, 127.1, 125.1, 121.4, 120.0, 83.0,79.2, 66.6, 57.7, 53.6, 47.2, 38.5, 31.2, 28.3, 28.1, 27.1. IR (CHCl₃) v(cm⁻¹) 3329, 3146, 3064, 2980, 1715, 1612, 1453, 1411, 1160.

Step 9: preparation of the carboxylic acid

Benzylic ester (10) (291 mg, 0.41 mmol) was dissolved in ethyl acetate(6 mL). Palladium on activated carbon (19 mg) was added, and thereaction mixture was put under an hydrogen atmosphere. The solutionmixture was stirred for 6 h, filtered on Celite®, rinsed with ethylacetate (5×10 mL) and concentrated. Aza-β-arginine was obtained as awhite foam (240 mg, 94%). ¹H NMR (300 MHz, CDCl₃) δ (ppm) 7.76-7.24 (m,8H), 4.49 (s, 2H, J=7.4 Hz), 4.21 (t, 1H, J=6.9 Hz), 3.68 (s, 2H), 3.49(s, 2H), 3.00 (s, 2H), 1.75 (s, 2H), 1.53-1.40 (m, 18H). ¹³C NMR (75.5MHz, CDCl₃) δ (ppm) 172.8, 156.8, 156.2, 153.0, 143.6, 141.3, 127.7,127.1, 125.1, 120.0, 83.7, 67.0, 58.8, 53.4, 47.2, 38.8, 28.2, 281,27.1. IR (CHCl₃) v (cm⁻¹) 3329, 2979, 1722, 1623, 1474, 1453, 1421,1231, 1150.

Example 2 Synthesis of Amino Acid Aza-β3-Leucine Step 1: Preparation ofFmoc-Hydrazine

Hydrazine (18.0 mL, 213 mmol) was dissolved in diethyl ether (240 mL) at0° C.A solution of Fmoc chloride (1) (12.0 g, 46.4 mmol) in diethylether (240 mL) was added to the hydrazine solution over a 30-minuteperiod. The reaction mixture was stirred at room temperature for 16 h.The solution was evaporated, and water (400 mL) and ethyl acetate (400mL) were added. The organic phase was washed with water (4×150 mL). Theresulting suspension was evaporated. Fmoc-hydrazine (2) was obtained asa white solid (13.92 g, 118%). ¹H NMR (300 MHz, CDCl₃) δ (ppm) 7.71-7.29(m, 8H), 6.05 (s, 1H), 4.45 (d, 1H, J=6.8 Hz), 4.23 (t, 1H, J=8.3 Hz),3.81 (s, 2H). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm) 143.6, 141.3, 127.8,127.1, 120.1, 67.3, 47.1 IR (CHCl₃) v (cm⁻¹) 1686, 1633, 1506, 1446.

Step 2: Preparation of the Imine

Fmoc-hydrazine (2) (3.66 g, 14.4 mmol) was dissolved in dichloromethane(55 mL), and isobutyraldehyde (1.31 mL, 14.4 mmol) was added. Themixture was stirred for 16 h and evaporated. The product (3) wasobtained as a white powder (4.12 g, 93%). ¹H NMR (300 MHz, CDCl₃) δ(ppm) 7.78-7.26 (m, 8H), 7.09 (d, 1Hm J=4.7 Hz), 4.51 (d, 2H, J=6.8 Hz),4.29 (s, 1H), 2.64 (sext, 1H, J=4.1 Hz), 1.13 (d, 6H, J=5.9 Hz). ¹³C NMR(75.5 MHz, CDCl₃) δ (ppm) 143.7, 141.3, 127.8, 127.1, 125.2, 120.0,67.2, 47.0, 31.4, 19.9. IR (CHCl₃) v (cm⁻¹) 3237, 3068, 1258, 2866,1708, 1545, 1464, 1450, 1382, 1354, 1259, 1185.

Step 3: Imine Reduction

The imine (3) (4.12 g, 13.4 mmol) was dissolved in 70 mL of a mixture ofdichloromethane and methanol (3:2). NaBH₃CN (1.01 g, 16.0 mmol) wasadded and the pH was brought up to 4 with 1N HCl. The reaction mixturewas stirred for 30 minutes. The solution was acidified to pH 1 with 1NHCl, and stirred for 10 minutes. The pH was then brought up to 7 withsolid sodium carbonate, and then evaporated. The residue was dissolvedin ethyl acetate (50 mL), and the organic phase was washed with water(1×50 mL) and brine (1×50 mL). The organic phase was dried with sodiumsulphate, filtered and evaporated. The crude product was purified with aflash chromatography on silica gel (eluent:ethyl acetate/hexanes 3:7).The reduced imine (4) was obtained as a white powder (4.63 g, 112%). ¹HNMR (300 MHz, CDCl₃) δ (ppm) 7.81-7.21 (m, 8H), 4.45 (s, 2H), 4.22 (t,1H, J=6.6 Hz), 2.90 (s, 2H), 1.97 (s, 1H), 0.98 (d, 6H, J=5.2 Hz). ¹³CNMR (75.5 MHz, CDCl₃) δ (ppm) 157.2, 143.7, 141.3, 127.8, 127.1, 125.0,120.0, 67.0, 60.0, 47.2, 26.7, 20.5. IR (CHCl₃) v (cm⁻¹) 3322, 3255,3064, 2955, 2884, 1694, 1527, 1489, 1457, 1383, 1273, 1192.

Step 4: Addition of Glyoxylic Acid

The reduced imine (4) (4.63 g, 14.9 mmol) was dissolved in 70 mL of amixture of dichloromethane and methanol (3:2). Glyoxylic acid (1.65 g,17.9 mmol) and NaBH₃CN (1.13 g, 17.9 mmol) were added. The pH wasbrought up to ph 4 with 1N HCl and stirred for 30 minutes, and themixture was acidified to pH 1 for 10 minutes. The pH was then brought upto 4 with solid sodium carbonate. The reaction mixture was filtered andconcentrated. The residue was dissolved in ethyl acetate (50 mL), andwashed with water (1×50 mL) and brine (1×50 mL). The organic phase wasdried with sodium sulphate, filtered and evaporated. Aza-β3-leucine (5)was obtained as a white solid foam (5.11 g, 93%). ¹H NMR (300 MHz,CDCl₃) δ (ppm) 7.76-7.31 (m, 8H), 6.12 (s, 1H), 4.54 (d, 2H, J=6.2 Hz),4.18 (s, 1H), 3.55 (s, 2H), 2.58 (d, 2H, J=7.0 Hz), 1.54 (s, 1H), 0.91(d, 6H, J=6.5 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ (ppm) 171.2, 157.2,143.4, 141.3, 127.8, 127.1, 124.9, 120.0, 67.1, 66.7, 60.5, 47.2, 26.3,20.5. IR (CHCl₃) v (cm⁻¹) 3251, 3051, 2958, 2869, 1739, 1514, 1451,1364, 1254, 1147.

Example 3 Synthesis of 4-amino-2-en-1-yl guanidine Step 1: preparationof 1-bromo-4-phthalimido-2-butene (1)

1,4-Dibromo-2-butene (15.0 g, 70.1 mmol) was added to the stirredsuspension of potassium phthalimide (4.32 g, 23.3 mmol) in DMF (24 mL).The mixture was stirred 48 h. Cooled water was added and theprecipitated solid was filtered and dried at high vacuum to give thedesired compound (1). The compound was purified with ethylacetate/hexane 3:7 to give a white solid (4.28 g, 66%); ¹H NMR (CDCl₃) δ(ppm) 3.92 (d, 2H, CH₂), 4.32 (d, 2H, CH₂), 5.89 (m, 2H, CH), 7.70-7.89(m, 4H, Aromatic); ¹³C NMR (CDCl₃) δ (ppm) 168 (CO), 134 (CH), 132 (Caromatic), 129 (C aromatic), 128 (C aromatic), 123 (CH), 38 (CH₂N), 31(CH₂Br).

Step 2: Preparation of N-(4-phthalimido-2-butenyl)hexamethylenetetrammonium bromide (2)

To a solution of 1-3 Hexamethylenetetramine (3.21 g, 22.9 mmol) in CHCl₃(43 mL) was added dropwised a solution of 1-bromo-4-phthalimido-2-butene(1) (4.28 g, 15.3 mmol) in CHCl₃ (43 mL), The solution was stirredduring 48 h. A white precipitate appeared. The solid was filtered andwashed with chloroform. A white powder was obtained (6.91 g, 107% watertrace, dried on high vacuum). ¹H NMR (MeOD) δ (ppm) 3.45 (d, 2H, CH₂),4.38 (d, 2H, CH₂), 4.50 (d, 6H, CH₂), 4.67 (d, 6H, CH₂), 5.85 (m, 1H,CH), 6.15 (m, 1H, CH), 7.80-7.89 (m, 4H, aromatic). ¹³C NMR (MeOD) δ(ppm) 168 (CO), 138 (C aromatic), 134 (C aromatic), 122 (CH), 117 (CH),78 (CH₂), 70 (CH₂), 57 (CH₂N), 38 (CH₂Br).

Step 3: preparation of N-(4-phthalimido-2-butenyl)ammonium chloride (3)

To a solution of compound (2) (4.32 g, 10.3 mmol) in ethanol (174 mL)was added dropwide a solution of concentrated HCl (7.25 mL, 12 M). Themixture was reflux during 2 h (around 90° C.). On cooling of thereaction mixture, the precipitate was filtered off and the filtrate wasconcentrated to give the desired product as a yellow oil (4.39 g). ¹HNMR (MeOD) δ (ppm) 3.50 (d, 2H, CH₂), 4.28 (d, 2H, CH₂), 5.75 (m, 1H,CH), 5.95 (m, 1H, CH), 7.79-7.87 (m, 4H, aromatic). ¹³C NMR (MeOD) δ(ppm) 168 (CO), 134 (C aromatic), 132 (C aromatic), 131 (C aromatic),124 (CH), 123 (CH), 40 (CH₂N), 38 (CH₂Br).

Step 4: preparation of N-(4-phthalimido-2-butenyl) guanidine-(di-Boc)(4)

To a solution of triethylamine (1.05 mL),tert-butyl[N-(tert-butoxycarbonyl)-N′-(trifluoroacetyl)carbamimidoyl]carbamate(6) (1.97 g, 5.03 mmol) in DCM (56 mL) was added compound (3) (1.4 g,5.55 mmol). The solution was stirred overnight. DCM was added and theorganic phase was washed with sodium bisulfate (2M), a saturatedsolution of NaHCO₃ and brine. The organic phase was dried with magnesiumsulfate, filtered and concentrated. The crude compound was purified withethyl acetate/hexane (30/70) to (40/60). Rf=0.58. A white solid wasobtained (2.13 g, 93%). ¹H NMR (CDCl₃) δ (ppm) 1.48 (s, 18H, CH₃), 4.05(m, 2H, CH₂), 4.28 (d, 2H, CH₂), 5.30 (m, 2H, CH), 7.73-7.84 (m, 4H,aromatic), 8.45 (sl, 1H, NH), 11.48 (s, 1H, NH). ¹³C NMR (CDCl₃) δ (ppm)133 (C aromatic), 132 (C aromatic), 129 (C aromatic), 126 (CH), 123(CH), 41 (CH₂), 38 (CH₂), 28 (CH₃ boc).

Step 5: preparation of N-(2-butenyl) guanidine-(di-Boc) (5)

To a solution of compound (4) (1.86 g, 4.06 mmol) in methanol (12.01 mL)and chloroform (9.5 mL) was added hydrazine (1.0 mL). The solution wasstirred during 4 h. A white solid appeared during the reaction. Thesolid was filtered and the filtrate was diluted with chloroform. Theorganic phase was washed with sodium hydroxide (1M). The organic phasewas dried with magnesium sulfate, filtered and concentrated. We obtaineda yellow solid (1.22 g, 92%). ¹H NMR (CDCl₃) δ (ppm) 1.49 (s, 18H, CH₃),3.30 (d, 2H, CH₂), 4.06 (d, 2H, CH₂), 5.65-5.76 (m, 2H, CH), 8.34 (s,1H, NH), 11.51 (s, 1H, NH). ¹³C NMR (CDCl₃) δ (ppm) 163 (CO), 155 (CO),153 (C), 134, 128, 125, 83, 79, 43 (CH₂), 28 (CH₃ boc).

Example 4 Synthesis of Aza-β3-Lysine Amino Acid Step 1: preparation oftert-butyl N-(4,4-diethoxybutyl)carbamate (1)

In a flask were added 4,4-diethoxybutan-1-amine (2 g, 12.4 mmol),triethylamine (1.8 mL, 12.9 mmol) and DCM (10 mL). The solution wascooled to 0° C. To this solution was added dropwised a solution of Boc₂O(2.7 g, 12.4 mmol) in DCM (10 mL). The solution was stirred overnightthen evaporated. The compound was purified by flash chromatography byusing ethyl acetate/hexane (10/90) to (30/70) to give (0.72 g, 89%). ¹HNMR (CDCl₃) δ (ppm) 1.17 (t, 6H, CH₃), 1.43 (s, 9H, CH₃), 1.55 (m, 4H,CH₂), 3.12 (d, 2H, CH₂), 3.45 (m, 2H, CH₂), 3.47 (m, 2H, CH₂), 4.47 (t,1H, CH), 4.64 (s, 1H, NH).

Step 2: preparation of N-t-butyloxycarbonyl-4-amino-butanal (2)

A solution of 1-Boc-amino-3,3-diethoxypropane (15.3 g, 58.5 mmol) inAcOH (27 mL) and water (8 mL) was stirred at room temperature for 10 h,neutralized with Na₂CO₃, taken up in ether, and washed with water andbrine. The organic phase was evaporated under vacuum to give a yellowoil used as such in the next step (14.25 g). ¹H NMR (400 MHz, CDCl₃) δ(ppm) 1.47 (s, CH₃ boc), 1.90 (m), 3.47 (m), 3.51 (q), 5.30 (s).

Step 3: preparation of Fmoc-NHN═CH(CH₂)₂NHBoc (3)

Fmoc carbazate (14.86 g, 58.4 mmol) was added to a stirred solution ofthe aldehyde (2) (10.95 g, 58.5 mmol) (261 mL) The reaction mixture wasstirred for 12 h at 45° C. and concentrated under vacuum to give crudesolid that was triturated with petroleum ether to afford the hydrazoneas a white solid (24.38 g). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.35 (s, 9H,CH₃ boc), 1.70 (m, 2H, CH₂), 2.10 (m, 2H, CH₂), 2.90 (m, 2H, CH₂), 4.20(m, 1H, CH), 6.83 (sl, 1H, NH), 7.65 (d, CH aromatic), 7.85 (d, CHaromatic), 8.66 (sl, 1H, NH), 10.70 (sl, 1H, NH).

Step 4: preparation of FmocNHN═CH(CH₂)₂NHBoc (4)

Then, Fmoc protected hydrazone (3) (24.38 g, 58.4 mmol) was dissolved ina mixture DCM/MeOH (166/100 mL) and was added NaBH₃CN (4.44 g, 70.7mmol). The pH was adjusted at pH 3-4 with HCl (2N) (Keep the pH at 3-4during 1 h). The solution was neutralized with NaHCO₃ (pH 7-8). Thesolvent was concentrated under vacuum. Ethyl acetate was added and theorganic phase was washed with water, brine and dried with magnesiumsulfate. The organic phase was filtered and concentrated then purifiedby flash chromatography by using dichloromethane/ethyl acetate 60/40 togive oil (4.46 g, 18%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.35 (s, 9H, CH₃boc), 2.62 (m, 2H, CH₂), 3.12 (m, 2H, CH₂), 3.78 (m, 2H, CH₂), 6.05 (sl,1H, NH), 6.36 (sl, 1H, NH), 7.27 (t, 1H, CH), 7.30 (t, 1H, CH), 7.65 (d,1H, CH), 7.85 (d, 1H, CH).

Step 5: preparation of FmocNHN(CH₂—COOH)—CH(CH₂)₂NHBoc (5)

To a solution of compound (2) (4.4 g, 10.50 mmol), glyoxylic acid (1.83g, 19.88 mmol) in a mixture of MeOH/DCM (60/30) was added NaBH₃CN (1.24g, 19.73 mmol). The pH was controlled between 3-4 by addition of HCl(2N) during one hour. The solution was filtrated and concentrated. Ethylacetate was added and the organic phase was washed with water and brine.The organic phase dried with magnesium sulfate and concentrated undervacuum to give the crude compound. The product was purified by flashchromatography (Ether(95)/MeOH(5)/AcOH(0.25) to give a white solid (4.1g, 82%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.35 (s, 9H, CH₃ boc), 2.62 (m,2H, CH₂), 3.12 (m, 2H, CH₂), 3.78 (m, 2H, CH₂), 6.05 (sl, 1H, NH), 7.27(t, 1H, CH), 7.30 (t, 1H, CH), 7.65 (d, 1H, CH), 7.85 (d, 1H, CH).

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention, and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth, and as follows in thescope of the appended claims.

1. A peptide sequence comprising the following formula IZ-Xaa₈-Xaa₇-Xaa₆-Xaa₅-Arg₄-Xaa₃-Xaa₂-Arg₁-Xaa_(1′)  (I) wherein Xaa_(1′)is absent or any amino acids, peptidomimetic, or stereoisomer thereof;Arg₁ and Arg₄ are independently arginine, an analogue of arginine orstereoisomer thereof; Xaa₂ is a basic amino acid, an analogue orstereoisomer thereof; Xaa₃ is independently any amino acids, an analogueor stereoisomer thereof; Xaa₅, Xaa₆, Xaa₇ and Xaa₈ independently areLys, Arg or His, peptidomimetic or stereoisomer thereof; and Z comprisesat least one of acetyl, azido and PEG group, fatty acids, steroidderivatives and sugars linked to the N-terminal of the peptide sequence;with the proviso that Xaa₅, Xaa₆, Xaa₇ and Xaa₈ are not aromatic ornegatively charged amino acids.
 2. The peptide sequence of claim 1wherein Xaa₂ is Lys, Arg or stereoisomer thereof and Xaa₃ isindependently Lys, Val or stereoisomer thereof.
 3. The peptide sequenceof claim 1, wherein at least one of Xaa_(1′), Arg₁, Arg₄, Xaa₅, Xaa₆,Xaa₇, and Xaa₈ is an analogue of Lys or Arg.
 4. The peptide sequence ofclaim 1, wherein the analogue of arginine is represented by thefollowing formula (III)

wherein W₁ is -alkyl-, -alkenylene-, -arylene-, -heteroarylene-,-arylalkylene-, -arylalkenylene-, —NR1-alkylene-, —NR1-alkenylene-,—NR1-arylene-, —NR1-heteroarylene-, —NR1-arylalkylene-,—NR1-arylalkenylene-, -alkylene-NR2-, -alkenylene-NR2-, -arylene-NR2-,-heteroarylene-NR2-, -arylalkylene-NR2-, -arylalkenylene-NR2-, —NR1alkylene-NR2-, —NR1-alkenylene-NR2-, —NR1-arylene-NR2-,—NR1-heteroarylene-NR2-, —NR1-arylalkylene-NR2-, or—NR1-arylalkenylene-NR2-; each of which may be optionally substitutedwith at least of one substituent selected from alkyl, alkenyl, aryl,heteroaryl, alkylene-COOH, alkenylene-COOH, arylene-COOH,heteroarylene-COOH, arylalkylene-COOH, and heteroarylalkylene-COOH; andR1 and R2 are independently H, alkyl, alkenyl, alkylene-COOH,alkenylene-COOH, arylene-COOH, heteroarylene-COOH, arylalkylene-COOH,heteroarylalkylene-COOH or alkenylcarboxy.
 5. The peptide sequence ofclaim 4, wherein W₁ is -arylalkylene-, -arylalkenylene-, —NR1-alkylene-,—NR1-alkenylene-, —NR1-arylene-, —NR1-arylalkylene-, -alkylene-NR2-,-alkenylene-NR2-, —NR1-alkenylene-NR2-, —NR1-arylene-NR2-, or—NR1-arylalkylene-NR2-.
 6. The peptide sequence of claim 4, wherein theanalogue of arginine is represented by


7. The peptide sequence of claim 1, wherein the analogue of Lys isrepresented by the following formula IV

wherein Y2 is N or CH; R10 is H, alkyl, alkenyl or alkyl-NR11-R12; R11and R12 are independently H or alkyl; W₂ is a bond, -alkylene-,-alkenylene-, -arylene-, -heteroarylene-, -arylalkylene-,-heteroarylalkylene-, -alkylarylene-, -alkylheteroarylene-,-alkenylarylene-, -alkenylheteroarylene-, each of which may beoptionally substituted with at least of one substituent selected fromalkyl, and alkenyl.
 8. The peptide sequence of claim 7, wherein W₂ is abond, -alkylene-, -alkenylene-, -heteroarylene-, -arylalkylene-,-heteroarylalkylene-, -alkylarylene-, or -alkylheteroarylene-, each ofwhich may be optionally substituted with at least of one substituentselected from alkyl, and alkenyl.
 9. The peptide of claim 7, wherein theanalogue of Lys is represented by


10. A composition comprising the peptide of claim 1 and a carrier.11.-16. (canceled)
 17. A method of reducing pathogen proliferation ortreating a pathogen infection in a subject, comprising administering thepeptide of claim 1 or the composition of claim 10 to the subject,thereby reducing the proliferation of the pathogen in the subject ortreating the pathogen infection in the subject.
 18. The method of claim17, wherein the pathogen is Anthrax, Pseudomonas, Botulism, Diphtheria,Aeromonas, Shigella, Influenzavirus A, parainfluenza, Sindbis virus,Newcastle disease virus, flavivirus, cytomegalovirus, herpesvirus, HIV,Measles virus, infectious bronchitis virus, Coronavirus, Marburg virus,Ebola virus or Epstein-Barr virus. 19.-27. (canceled)
 28. The method ofclaim 17, further comprising administering at least one anti-viral drug.29. The method of claim 17, wherein said peptide or composition isadministered concurrently with at least one anti-viral drug.
 30. Themethod of claim 17, wherein said peptide or composition is administeredby one of the following routes: oral, mucosal, intranasal, intraocular,intratracheal, intrabronchial, intrapleural, intraperitoneal,intracranial, intramuscular, intravenous, intraarterial, intralymphatic,subcutaneous, intratumoral, gastric, enteral, colonic, rectal, urethralor intravesical.